Efficient Binding of 4/7 α-Conotoxins to Nicotinic α4β2 Receptors Is Prevented by Arg185 and Pro195 in the α4 Subunit

Abstract

␣-Conotoxins are subtype-selective nicotinic acetylcholine receptor (nAChR) antagonists. Although potent ␣3␤2 nAChRselective ␣-conotoxins have been identified, currently characterized ␣-conotoxins show no or only weak affinity for ␣4␤2 nAChRs, which are, besides ␣7 receptors, the most abundant nAChRs in the mammalian brain. To identify the determinants responsible for this difference, we substituted selected amino acid residues in the ligand-binding domain of the ␣4 subunit by the corresponding residues in the ␣3 subunit. Two-electrode voltage clamp analysis of these mutants revealed increased affinity of ␣-conotoxins MII, TxIA, and [A10L]TxIA at the ␣4(R185I)␤2 receptor. Conversely, ␣-conotoxin potency was reduced at the reverse ␣3(I186R)␤2 mutant. Replacement of ␣4Arg185 by alanine, glutamate, and lysine demonstrated that a positive charge in this position prevents ␣-conotoxin binding. Combination of the R185I mutation with a P195Q mutation outside the binding site but in loop C completely transferred high ␣-conotoxin potency to the ␣4␤2 receptor. Molecular dynamics simulations of homology models with docked ␣-conotoxin indicate that these residues control access to the ␣-conotoxin binding site.

α‐Conotoxins active at α3‐containing nicotinic acetylcholine receptors and their molecular determinants for selective inhibition

“…Mutagenesis experiments and molecular dynamic (MD) simulations showed that the residue exchange causes structural changes at the ACh binding site by reducing the flexibility of nearby receptor residues and preventing them from effective interaction with RegIIA residues due to steric hindrance (Kompella et al, ). A similar effect has been reported for α4β2, whereby the reverse mutation at the homologous position in rat α4, P195Q, enhanced α‐conotoxin TxIA potency at α4β2 by increasing the number of contacts between toxin and receptor (Beissner et al, ).…”

Design and synthesis of α‐conotoxin GID analogues as selective α4β2 nicotinic acetylcholine receptor antagonists

“…This stretch of the loop (187–200) is largely conserved between the α4 and α3‐subunits (see Supporting Information, Figure S1). It should be noted that R186, which has been shown to be important for α4β2 binding is also at the base of this loop …”

Positional Scanning Mutagenesis of α-Conotoxin PeIA Identifies Critical Residues That Confer Potency and Selectivity for α6/α3β2β3 and α3β2 Nicotinic Acetylcholine Receptors

“…Other residues of the C-loop have been shown to play a more direct role in the binding of ␣-Ctxs. Beissner et al (37) found that an Arg in position 185 of the ␣4 subunit prevents high affinity binding of several 4/7 ␣-Ctxs to the ␣4␤2 subtype, and mutation to Ile, the residue found in the homologous position of ␣6 and ␣3 subunits, increased binding affinity. Last, the differing residues of the various ␣ subunits may produce unique conformations of the C-loop itself, allowing ␣-Ctxs to bind to some receptor subtypes but not others, as suggested in a recent study examining the binding of ␣-Ctx BuIA to ␣6/␣3␤2␤3 nAChRs (38).…”